Dataset Information

Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and in vivo Subcellular Location.

ABSTRACT: Type IV Coupling Proteins (T4CPs) are essential elements in many type IV secretion systems (T4SSs). The members of this family display sequence, length, and domain architecture heterogeneity, being the conserved Nucleotide-Binding Domain the motif that defines them. In addition, most T4CPs contain a Transmembrane Domain (TMD) in the amino end and an All-Alpha Domain facing the cytoplasm. Additionally, a few T4CPs present a variable domain at the carboxyl end. The structural paradigm of this family is TrwBR388, the T4CP of conjugative plasmid R388. This protein has been widely studied, in particular the role of the TMD on the different characteristics of TrwBR388. To gain knowledge about T4CPs and their TMD, in this work a chimeric protein containing the TMD of TraJpKM101 and the cytosolic domain of TrwBR388 has been constructed. Additionally, one of the few T4CPs of mobilizable plasmids, MobBCloDF13 of mobilizable plasmid CloDF13, together with its TMD-less mutant MobB?TMD have been studied. Mating studies showed that the chimeric protein is functional in vivo and that it exerted negative dominance against the native proteins TrwBR388 and TraJpKM101. Also, it was observed that the TMD of MobBCloDF13 is essential for the mobilization of CloDF13 plasmid. Analysis of the secondary structure components showed that the presence of a heterologous TMD alters the structure of the cytosolic domain in the chimeric protein. On the contrary, the absence of the TMD in MobBCloDF13 does not affect the secondary structure of its cytosolic domain. Subcellular localization studies showed that T4CPs have a unipolar or bipolar location, which is enhanced by the presence of the remaining proteins of the conjugative system. Unlike what has been described for TrwBR388, the TMD is not an essential element for the polar location of MobBCloDF13. The main conclusion is that the characteristics described for the paradigmatic TrwBR388 T4CP should not be ascribed to the whole T4CP family. Specifically, it has been proven that the mobilizable plasmid-related MobBCloDF13 presents different characteristics regarding the role of its TMD. This work will contribute to better understand the T4CP family, a key element in bacterial conjugation, the main mechanism responsible for antibiotic resistance spread.

SUBMITTER: Alvarez-Rodriguez I

PROVIDER: S-EPMC7431656 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and <i>in vivo</i> Subcellular Location.

Álvarez-Rodríguez Itxaso I Ugarte-Uribe Begoña B de la Arada Igor I Arrondo José Luis R JLR Garbisu Carlos C Alkorta Itziar I

Frontiers in molecular biosciences 20200811

Type IV Coupling Proteins (T4CPs) are essential elements in many type IV secretion systems (T4SSs). The members of this family display sequence, length, and domain architecture heterogeneity, being the conserved Nucleotide-Binding Domain the motif that defines them. In addition, most T4CPs contain a Transmembrane Domain (TMD) in the amino end and an All-Alpha Domain facing the cytoplasm. Additionally, a few T4CPs present a variable domain at the carboxyl end. The structural paradigm of this fami ...[more]

PMID: 32850972

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Project description:BACKGROUND: In the past decades, various protein subcellular-location (SCL) predictors have been developed. Most of these predictors, like TMHMM 2.0, SignalP 3.0, PrediSi and Phobius, aim at the identification of one or a few SCLs, whereas others such as CELLO and Psortb.v.2.0 aim at a broader classification. Although these tools and pipelines can achieve a high precision in the accurate prediction of signal peptides and transmembrane helices, they have a much lower accuracy when other sequence characteristics are concerned. For instance, it proved notoriously difficult to identify the fate of proteins carrying a putative type I signal peptidase (SPIase) cleavage site, as many of those proteins are retained in the cell membrane as N-terminally anchored membrane proteins. Moreover, most of the SCL classifiers are based on the classification of the Swiss-Prot database and consequently inherited the inconsistency of that SCL classification. As accurate and detailed SCL prediction on a genome scale is highly desired by experimental researchers, we decided to construct a new SCL prediction pipeline: LocateP. RESULTS: LocateP combines many of the existing high-precision SCL identifiers with our own newly developed identifiers for specific SCLs. The LocateP pipeline was designed such that it mimics protein targeting and secretion processes. It distinguishes 7 different SCLs within Gram-positive bacteria: intracellular, multi-transmembrane, N-terminally membrane anchored, C-terminally membrane anchored, lipid-anchored, LPxTG-type cell-wall anchored, and secreted/released proteins. Moreover, it distinguishes pathways for Sec- or Tat-dependent secretion and alternative secretion of bacteriocin-like proteins. The pipeline was tested on data sets extracted from literature, including experimental proteomics studies. The tests showed that LocateP performs as well as, or even slightly better than other SCL predictors for some locations and outperforms current tools especially where the N-terminally anchored and the SPIase-cleaved secreted proteins are concerned. Overall, the accuracy of LocateP was always higher than 90%. LocateP was then used to predict the SCLs of all proteins encoded by completed Gram-positive bacterial genomes. The results are stored in the database LocateP-DB http://www.cmbi.ru.nl/locatep-db1. CONCLUSION: LocateP is by far the most accurate and detailed protein SCL predictor for Gram-positive bacteria currently available.

Dataset Information

Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and in vivo Subcellular Location.

Publications

Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and <i>in vivo</i> Subcellular Location.

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